Computational protocol: Primary Spinal OPC Culture System from Adult Zebrafish to Study Oligodendrocyte Differentiation In Vitro

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Protocol publication

[…] For the RNAseq experiment, the SMARTer Ultra Low Input RNA for Illumina Sequencing Kit - HV from Clontech was used to reverse transcribe the RNA and amplify full-length cDNA according to the user manual. Therefore, 2000 cells from five biological replicates of both lines Tg(olig2:eGFP) and Tg(mbp:eGFP) were directly FACsorted in 5 μL reaction buffer containing RNAse Inhibitor. After amplification of cDNA using 12 cycles, the cDNA was sheared to 200 bp fragment length with 8-micro TUBE strip in the Ultrasonicator Covaris LE220, followed by a library prep using NEBNext Ultra DNA Library Prep Kit for Illumina (NEB). The libraries were sequenced with 75 bp single end on a Hiseq2500 with a minimal sequencing depth of 27 million reads and mapped to GRCz10 with GSNAP (v 2016-09-23). Transcripts were counted with feature Counts v 1.5.2 based on Ensembl version 81. The gene expression values were normalized by the R-package DESeq2 in R (v 3.3.0). […]

Pipeline specifications

Software tools GSNAP, DESeq2
Databases Full Length cDNA
Application RNA-seq analysis
Organisms Danio rerio, Homo sapiens
Diseases Demyelinating Diseases, Spinal Cord Injuries, Wounds and Injuries, Chemical and Drug Induced Liver Injury
Chemicals Lysophosphatidylcholines